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May 27, 1992 - University of Waterloo, Waterloo, Ontario, Canada N1G 2G1 .... tomato (Lycopersicon esculentum L.) membrane-associated li-. 1802 ...

Received for publication May 27, 1992 Accepted September 13, 1992

Plant Physiol. (1992) 100, 1802-1807 0032-0889/92/100/1 802/06/$01 .00/0

Purification and Partial Characterization of a Membrane-Associated Lipoxygenase in Tomato Fruit' Caroline G. Bowsher2, Bonita J. M. Ferrie, Sibdas Ghosh, James Todd3, John E. Thompson, and Steven J. Rothstein* Department of Molecular Biology and Genetics (C.G.B., B.J.M.F., S.J.R.) and Department of Horticultural Science (J.T., J.E.T.), University of Guelph, Guelph, Ontario, Canada NIG 2W1; and Department of Biology (S.G., I.E.T.), University of Waterloo, Waterloo, Ontario, Canada N1G 2G1 ABSTRACT

activity has been recognized for more than 50 years (1), and it has also been termed carotene oxidase, fat oxidase, and lipoxidase. This notwithstanding, it has proven difficult to assign definitive physiological roles to lipoxygenase. A number of different roles have been postulated for lipoxygenase in plant growth and development. However, the only role for which evidence appears to be unambiguous is that of a vegetative storage protein in soybean (22). The lipoxygenase reaction results in the formation of a hydroperoxide product that is a highly reactive species (23). Furthermore, there is evidence that this reaction also leads to the production of superoxide radical (10). The generation of these highly reactive species has promoted the hypothesis that lipoxygenase plays a role in cell senescence (19) and in pathogen defense (9, 18). It also has been proposed that lipoxygenase may be involved in membrane turnover (18) and in various other cellular and developmental processes (for reviews, see refs. 9 and 18). The subcellular localization of lipoxygenase in different plant tissues has been examined in a number of studies (reviewed in refs. 11 and 23). Nearly every subcellular compartment of higher plant cells has been implicated as a site of lipoxygenase localization, and it has been pointed out that the enzyme may adsorb nonspecifically to membranous fractions (11). Most of the evidence suggests that lipoxygenase is predominantly a soluble, cytoplasmic enzyme. However, there have been reports of it being detected in the stroma and thylakoids of chloroplasts (4), in a mitochondrial fraction (7), and in vacuoles (25). Recently, a membranous lipoxygenase that was tentatively assigned to the nuclear membrane was isolated from tulip bulbs (14). Although precise localizations of lipoxygenase in plant cells remain unclear, it has been shown in mammalian cells that a soluble form of the enzyme is translocated to membranes upon the influx of extracellular Ca2" and can then metabolize substrates released by membrane phospholipases (17, 26). We are interested in the potential role of lipoxygenase in plant senescence and membrane turnover. It can be hypothesized that for lipoxygenase to play an important role in these processes it should be associated with cell membranes; yet, the best characterized plant lipoxygenases are soluble. Recently, a membrane-associated lipoxygenase activity was found in tomato fruit (20) and senescing carnation petals (15). In this paper, we describe the purification of the green tomato (Lycopersicon esculentum L.) membrane-associated li-

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 ,mol *min' . mg-' of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent Km of 6.2 Mm, and Vm,x of 10.3 jAmol-min-' mg-' of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 Mm and 1.3 Amol min-'.mg-' of protein, respectively. Thus, the membraneassociated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.

The predominant mechanism for fatty acid catabolism is fl-oxidation, which occurs in mitochondria or glyoxysomes. Although this pathway metabolizes most of the fatty acids derived from stored fats and oils, several other routes are utilized for the oxidation of specialized fatty acids. One of these alternative pathways involves the enzyme lipoxygenase (EC 1.13.11.12), which catalyzes the oxidation of a class of unsaturated fatty acids having a cis,cis-1,4-pentadiene structure. These fatty acids are primarily present as constituents of membrane phospholipids. The existence of this enzyme 'This work was supported by grants to S.J.R. and J.E.T. from the Natural Sciences and Engineering Research Council of Canada. 2 Present address: Plant Metabolism Research Unit, Department of Cell and Structural Biology, University of Manchester, Williamson Building, Oxford Road, Manchester, M13 9PL, England. 3Present address: Department of Biology, Queens University, Kingston, Ontario, Canada K7L 3N6.

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PURIFICATION OF TOMATO MEMBRANE-ASSOCIATED LIPOXYGENASE

poxygenase and compare its activity to that of the partially purified, soluble form of the enzyme. Membrane fractionation studies indicate that the predominant form of this membranous lipoxygenase is associated with the thylakoid membrane. MATERIALS AND METHODS

Lipoxygenase Purification The following buffers were used in the purification procedures. Buffer 1 (for extraction) was 100 mm Mops (pH 7.6), 10 mM EGTA, 7% (w/v) sucrose, and 1 mnm PMSF. Buffer 2 (for dialysis and chromatography) was 20 mm Tris-HCl (pH 7.2), 10% (v/v) glycerol, and 0.5% (v/v) Triton X-100. Buffer 3 (for dialysis and chromatography) was 20 mm Tris-HCl (pH 7.2) and 10% (v/v) glycerol. Pericarp tissue from green, breaker stage tomato (Lycopersicon esculentum L. cv Caruso) fruit was cut into small pieces of approximately 1 cm3 and either used immediately or stored at -200C. Tissue (400 g) was homogenized in 360 mL of buffer 1 using a Waring blender. The homogenate was filtered through four layers of cheesecloth and centrifuged at 7840g for 20 min in a refrigerated centrifuge. The supematant fluid was centrifuged at 180,000g for 50 min to yield a pellet of microsomal membranes and a soluble fraction (cytosol). The microsomal pellet was solubilized by resuspension in 40 mL of buffer 3 containing 2% (v/v) Triton X-100 and constant stirring for 2 h at 40C. This was then dialyzed overnight against buffer 2. The dialyzed fraction was clarified by centrifugation at 9250g for 10 min and loaded at 30 mL. h-1 onto a column (10 x 2.5 cm, i.d.) of DEAE-cellulose equilibrated with buffer 2. The column was washed with buffer 2 until no additional protein was eluted. Lipoxygenase activity was eluted from the column with a 400-mL linear gradient of 0 to 0.4 M NaCl in buffer 2. Active fractions were combined and applied at 20 mL h-1 to a gel filtration column (100 x 2.6 cm. i.d.) of Sephadex G-150 equilibrated with buffer 2, with 5-mL fractions collected. Lipoxygenase activity eluted as a single peak. Active fractions were combined and loaded at 20 mL- h-1 onto a column (6 x 1 cm, i.d.) of DEAESephacel (Pharmacia) equilibrated with buffer 2. Lipoxygenase activity was eluted from the column with a linear gradient of 0 to 0.4 M NaCl in buffer 2. The cytosolic fraction from the tomato fruit extract was made 10% (v/v) in glycerol, and ammonium sulfate was added to 25% saturation (40C). After standing for 1 h, the solution was centrifuged at 16,000g for 30 min. The precipitate was discarded, and solid, chilled ammonium sulfate was added to the supernatant fluid to a final saturation of about 60%. The precipitate was removed by centrifugation and dissolved in a minimum volume of buffer 3. The solution was clarified by centrifugation at 9250g for 10 min and dialyzed overnight against buffer 3. The dialyzed fraction was then applied to a DEAE-cellulose column equilibrated in buffer 3. Active fractions were eluted and sequentially applied to Sephadex G-150 and DEAE-Sephacel columns as described for the microsomal fraction, except that buffer 3 was used in all cases. -

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Lipoxygenase Activity and Protein Determination Lipoxygenase activity was determined spectrophotometrically at 234 nm by a standard assay that involves the measurement of conjugated diene formation using linoleic acid as the substrate at pH 7.0 and 220C (20). To monitor fractions eluted from the chromatography columns, a rapid qualitative iodometric spot assay for lipoxygenase activity was used (24). Protein concentration was determined as described by Bradford (2) using a system and dye reagent developed by BioRad. Alternatively, the Bio-Rad DC protein assay system was used.

Membrane Fractionation Microsomal membranes were washed by resuspension in buffer 1 and centrifugation at 180,000g for 60 min and then resuspended in 25 mm Mops-KOH (pH 7.0), at a concentration of 7.5 mg of protein.mL-1. Sucrose density gradient fractionation of the microsomal membranes and measurements of marker enzyme activities (vanadate-sensitive ATPase for plasma membrane and rotenone-insensitive NADHCyt c reductase for endoplasmic reticulum) were carried out as described previously (12). Chl was quantified by A652, and carotenoids were measured as described by Britton (3).

Thylakoid Isolation Thylakoid membranes were isolated from intact chloroplasts obtained from either mature leaves of tomato plants or pericarp of mature green tomato fruit. Intact chloroplasts were prepared according to the method of Pardo et al. (13). Briefly, a crude chloroplast pellet was obtained from leaves or pericarp by grinding gently in extraction buffer (1 g/5 mL) containing 100 mM Mops-KOH (pH 7.0), 5 mM MgC12, 300 mm sucrose, 2% (v/v) glycerol, and 1 mm PMSF. The homogenate was subjected to differential centrifugation (1 min at 150g; supernatant spun 2 min at 2000g) to obtain a partially purified chloroplast fraction, and purified intact chloroplasts were obtained by centrifugation of this fraction through a cushion of 6 M sucrose (15 min at 500g). Thylakoid membranes were obtained by rupturing the chloroplasts in hypotonic buffer (10 mm Mops-KOH [pH 7.0], 5 mM MgC12, and 1 mm PMSF) for 15 min on ice and centrifugation for 2 min at 2000g in a Sorvall HB-4 rotor. The isolated thylakoids were washed three times with hypotonic buffer, resuspended in 2 mL of SDS-PAGE sample buffer (62.5 mm Hepes-KOH [pH 6.8], 1 mm Na2EDTA, 10% [v/v] glycerol, 5% [v/v] 2mercaptoethanol, 3% SDS, and 1 mm PMSF), incubated for 3 min in a boiling water bath, cooled, and then centrifuged for 1 min at 14,000 rpm in an Eppendorf centrifuge (model 5415, Brinkmann Instruments). The supematant fraction was analyzed for protein (6) with BSA (fraction V, Sigma) as a standard.

Denaturing Electrophoresis SDS-PAGE of purified proteins was on 7.5% acrylamide gels according to the method of Hames (8). The gels were stained for protein with silver (28). Westem blotting was based on the method of Towbin et al. (21). Lipoxygenase

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BOWSHER ET AL.

Plant Physiol. Vol. 100, 1992

protein was visualized immunologically on western blots using chicken anti-soybean lipoxygenase 1 (20). Thylakoid proteins were fractionated on 12% SDS polyacrylamide gels.

Table II. Purification of Soluble Lipoxygenase Stage .o.l Protein Specific

RESULTS

timol/min

Purification of Lipoxygenase Lipoxygenase activity is highest in green, breaker stage tomatoes, and, hence, microsomal and cytosolic fractions from fruit at this stage of development were used for protein purification. Results of initial experiments indicated that membrane-associated lipoxygenase activity was extremely unstable. A number of conditions were tested, and it was found that having 10% (v/v) glycerol in the buffer was essential for stabilization of membranous lipoxygenase, thus allowing purification. The microsomal and soluble lipoxygenases were purified as summarized in Tables I and II, respectively. Approximately one-third of the total lipoxygenase activity was in the isolated microsomal membrane fraction in all of the purifications attempted. The lipoxygenase-specific activity of the membrane fraction was more than twice that of the corresponding, but less pure, soluble (cytosolic) fraction. The membrane lipoxygenase was purified approximately 50-fold to a final specific activity of 8.3 umol -min-' -mg-' of protein (Table I). It should be noted that the actual degree of purification might have been considerably higher given the instability of this enzyme. When the same protocol was used but with an ammonium sulfate precipitation carried out before column chromatography, a soluble enzyme was purified about 6-fold to 1.07 ,mol . min-1. mg-' of protein (Table II). The purified membrane lipoxygenase consisted primarily of a single band of 97-kD polypeptide when analyzed by SDS-PAGE and silver staining, although it was not completely free of contaminating proteins (Fig. 1). This 97-kD protein reacted with soybean lipoxygenase 1 antibody after electroblotting onto nitrocellulose (20) and was recognized at each step of the purification protocol (data not shown). In contrast, there were a number of contaminating proteins in the soluble lipoxygenase preparation, and it was not purified to near homogeneity by our standard protocol. The apparent native molecular mass of the membrane lipoxygenase was determined using a Sephadex G-150 column and was found to be 102 kD. The soluble lipoxygenase had an apparent molecular mass of 91 kD using this same criterion, even

Activity

Purification

mg

mg' protein

fold

37.6 22.0

222 175

0.17 0.13

1.0 0.8

20.1

91

0.22

1.3

14.8 12.4 10.7

55 37 10

0.27 0.33 1.07

1.6 1.9 6.3

Activity

Crude extract 180,000g supernatant

25-60% (N H4)2SO4 DEAE-cellulose Sephadex G-150

DEAE-Sephacel

though on an SDS-PAGE gel it had a similar mobility to that of the membrane enzyme. On elution of these enzymes from the anion-exchange DEAE-Sephacel column, the membrane lipoxygenase eluted at approximately 0.15 M NaCl in a single, sharp peak, whereas the soluble lipoxygenase eluted as a single, sharp peak at 0.2 M NaCl (data not shown). The purified lipoxygenase enzymes were analyzed both for their pH optimum and for specific kinetic parameters. The membrane and soluble forms both behaved in approximately the same fashion to varying pH, with an optimum at pH 6.5. Lineweaver-Burk plots with linoleic acid as the substrate gave an apparent Km value of 6.2 ,uM for the membrane enzyme and 3.8 ,uM for the soluble lipoxygenase (Fig. 2). The Vmax was 10.3 /Amol min-1-mg1' of protein for the membrane lipoxygenase and 1.3 gmol-min1-.mg-1 of protein for the soluble form of the enzyme. These values differ substantially from those reported for membrane-associated lipoxygenase by Todd et al. (20). There are two reasons for this discrepancy: first, in that work, crude microsomal protein was used for the assay; second, an incorrect extinction coefficient was used for the diene product of this reaction.

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Table I. Purification of Microsomal Lipoxygenase Stage

Crude extract 180,000g pellet DEAE-cellulose Sephadex G-

Activity

Total

Protein

Specific

Activity

Purification

,umol/min

mg

mg of protein

fold

0.17 0.31 0.76

1.0 1.8 4.5

8.3

49.0

37.6 12.6 8.4 6.8

222 41 11

45

N.D.a

150

DEAE-Sephacel 5.0 a Not determined.

66

0.6

Figure 1. SDS-PAGE of purified membrane-associated tomato lipoxygenase. Lane 1, SDS molecular mass markers with mass indicated in kD; lane 2, membrane-associated lipoxygenase eluted from the DEAE-Sephacel column. The gel was silver stained.

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and the solubilization of this activity required the use of Triton X- 100. Second, the membrane-associated lipoxygenase eluted from anion-exchange columns at a different ionic strength than did the soluble form. Third, work with purified chloroplasts (Fig. 4) indicates that lipoxygenase protein is associated with thylakoid membranes. Furthermore, in four different experiments, the amount of lipoxygenase activity that cosedimented with thylakoids was very similar (Fig. 3). Finally, the kinetic properties of the membrane-associated

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Localization of Membrane Lipoxygenase Washed microsomal membranes were fractionated on a linear sucrose gradient in an attempt to localize the membrane-associated lipoxygenase activity. The distributions of protein, lipoxygenase activity, carotenoids, Chl (a marker for thylakoids), vanadate-sensitive ATPase (a marker for plasma membrane), and rotenone-sensitive NADH-Cyt c reductase (a marker for ER) across the gradient were compared (Fig. 3). The bulk of the protein and lipoxygenase activity was associated with the thylakoid membranes. Indeed, the distribution profiles across the gradient for protein, lipoxygenase, and Chl were closely parallel, showing a major peak in fractions 11 to 16 (Fig. 3A). The distribution profiles for Chl and carotenoids were virtually identical (Fig. 3B), whereas the peaks of lipoxygenase, vanadate-sensitive ATPase, and rotenone-insensitive Cyt c reductase activities in the gradient were clearly distinguishable (Fig. 3C). Thus, the major portion of lipoxygenase activity in the microsomal fraction isolated from the green fruit appears to be associated with thylakoid membranes. Identification of lipoxygenase protein in thylakoid membranes was confirmed immunologically. Thylakoids obtained from intact chloroplasts isolated from either tomato leaves or mature, green fruit contain a protein at an apparent molecular mass of 97 kD that cross-reacted with polyclonal antibodies raised against soybean lipoxygenase 1 (Fig. 4). There was also an additional cross-reacting polypeptide at 32 kD (Fig. 4) that may be a lipoxygenase breakdown product. DISCUSSION In the present work, a membrane-associated lipoxygenase was purified approximately 50-fold from green breaker stage tomato fruit. Several lines of evidence indicate that this enzyme is truly associated with the membrane fraction and is not simply adsorbed nonspecifically to membranes during the isolation procedure. First, in five different enzyme preparations, approximately the same percentage (33%) of total lipoxygenase activity sedimented with the membrane pellet,

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